Enamelin is critical for enamel formation. In the absence of enamelin no true enamel is formed leading to the disorder of amelogenesis imperfecta in humans. In this proposal we focus our efforts on enamelin function in vivo. We hypothesize that 1) enamelin regulates the number of enamel crystals, 2) enamelin regulates the thickness of enamel mineral ribbons, 3) enamelin is a multifunctional protein and expression of critical parts of enamelin will partially recover the enamel phenotype in Enam null mice, 4) cysteines in the enamelin C-terminal domain are critical for enamelin function, and 5) enamelin provides feedback to ameloblasts that influences levels of matrix protein expression. Four specific aims are proposed to test our hypotheses. SA1: To determine the functional consequences of altered enamelin expression on crystal number and shape. SA2: To establish a transgenic system to assay the function of enamel proteins in vivo and use it to determine the functions of the enamelin N-terminal/32kDa and C-terminal cysteine-rich domains. SA3: To discern whether reduced enamelin or cell pathology alters ameloblastin and/or amelogenin expression. SA4: To express and purify recombinant enamelin for in vitro functional analyses. Our objectives are 1) to understand the biological actions of enamelin and its physiologic roles in dental enamel formation, 2) to dissect enamelin protein function using mouse models expressing specific enamelin domains, and 3) to express and purify recombinant enamelin for functional analyses. Our long-term goal is to understand the mechanism of enamel formation in vivo and to engineer enamel formation in vitro.